Production of homogeneous cell line highly permissive to porcine circovirus type 2 (PCV2) infection

ABSTRACT

Continuous cell lines that are highly permissive to infection by porcine circovirus type 2 (“PCV2”) are described. PCV2 is the causal agent of post-weaning multi-systemic wasting syndrome (“PMWS”) in pigs. PMWS has emerged as a major disease that poses a significant threat to the economics of global swine industry. The highly permissive cell lines of this invention provide efficient and reliable sources of PCV2 for use in development of vaccines, therapies and diagnostic agents for PMWS.

This application is a filing under 35 USC §371 of PCT/SG2007/000133,filed May 11, 2007. This application is incorporated herein byreference.

BACKGROUND OF THE INVENTION

The present invention relates to the production of porcine circovirustype 2 (PCV2). More particularly, the invention relates to continuouscell lines that are highly susceptible to infection with PCV2 and tomethods for the production of PCV2 using the cell lines.

Porcine circovirus (PCV) is a small, non-enveloped, circular,single-stranded DNA virus classified in the Circoviridae family. Murphy,F A., Fauquet, C M., Bishop, D H L., Ghabrial, S A., Jarvis, A W.,Martelli, G P.; Mayo, M A., Summers, M D. Virus taxonomy. Sixth reportof the International Committee on Taxonomy of Viruses. New York, N.Y:Springer-Verlag; 1995. pp. 166-168. It was originally identified anddescribed as a contaminant of a porcine kidney cell line. Tischer, I.,Gelderblom, H., Vettermann, W., Koch, M. A. A very small porcine viruswith circular single-stranded DNA. Nature. 1982:295:64-66. Recently, PCVhas been associated with a disease of pigs, the post-weaningmulti-systemic wasting syndrome (PMWS), first observed in WesternCanada. Ellis, J., Hassard, L., Clark, E., Harding, J., Allan, G.,Willson, P., Strokappe, J., Martin, K., McNeilly, F., Meehan, F., Todd,D., Haines, D. Isolation of circovirus from lesions of pigs withpostweaning multisystemic wasting syndrome. Can. Vet. J., 1998;39:44-51; Harding, J. C. S., Clark, E. G. Recognizing and diagnosingpostweaning multisystemic wasting syndrome (PMWS). Swine Health Prod.1997; 5:201-203; Jue Liu, Isabelle Chen, and Jimmy Kwang, J. Virol.2005: 79(13); 8262-74. PWMS has emerged as a major disease that poses asignificant threat to the economics of the global swine industry. Afterits first appearance in Canada, PMWS has now spread to the UnitedStates, Europe and Asia. The syndrome mainly affects pigs between 6 and14 weeks of age. It tends to be slow and progressive with a highfatality rate in affected pigs. See http:www dot aphis dot usda dotgov/vs/ceah backslash dei/taf backslashemergingdiseasenotice_files/pmws_(—)0301.htm.

The clinical signs of PMWS are quite variable. Affected pigs may showsigns of chronic wasting, respiratory distress, diarrhea,incoordination, paralysis, pale skin color and blue ears. Pigs usuallydemonstrate a decrease in growth rate and, occasionally, jaundice.

The diagnosis of PMWS is based on the age of affected pigs, typicalwasting appearance and necropsy lesions. Microscopic andimmunohistochemical examination of tissues reveals unique lung andlymphoid tissue lesions with the presence of PCV2. Id.

Antibacterial medication is usually ineffective in treating PWMS andcurrently no vaccines are available. Prevention of the syndrome is basedon biosecurity precautions and good husbandry practices.

PCV2 has also been found in association with other diseases includingporcine dermatitis and nephropathy syndrome (“PDNS”), congenital tremors(CT-All) reproductive disorders, prenatal myocarditis and proliferativeand necrotizing pneumonia.

Vaccines employing PCV2 antigens have shown some initial success inpreventing the PMWS. Fenaux, M., et al., A chimeric porcine circovirus(PCV) with the immunogenic capsid gene of the pathogenic PCV type 2(PCV2) cloned into the genomic backbone of the nonpathogenic PCV1induces protective immunity against PCV2 infection in pigs. J. Virol.,2004. 78(12): p. 6297-303; Blanchard, P. et al., Protection contre lamaladie d'amaigrissement du porcelet (MAP) par vaccins a ADNet proteinesrecombinantes. Journees de la Recherche Porcine en France, 2004. 36: p.345-352; Blanchard, P., et al., Protection of swine against porcinemultisystemic wasting syndrome (PMWS) by porcine circovirus type 2(PCV2) proteins. Vaccine, 2003. 21: p. 4565-4575; Pogranichniy, R. etal. Efficacy of inactivated PCV2 vaccines for preventing PMWS in CDCDpigs. American Association of Swine Veterinarians. 2004. Des Moines,Iowa. However, an effective vaccine is not currently available.

The development of vaccines, diagnostic agents and therapies for PMWSand other diseases associated with PCV2 viral infections will requireefficient and reliable means for producing the virus in substantialquantities. PCV2 virus stocks have conventionally been produced byculturing the virus in porcine kidney cell-line PK15. The virus titersyielded from PK15 cell cultures, expressed as 50% tissue cultureinfectious dosage (“TCID₅₀”) per milliliter, usually ranged from 10⁴-10⁵and could never exceed 10⁵. Immunofluorescence stainings of infectedPK15 cell cultures have revealed that only about 40% of the cellpopulation is susceptible to the PCV2 infection.

A need exists for a continuous cell line that is highly permissive toPCV2 infection and that reliably produces virus in high titers overextended periods of time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows immunofluorescence assay results demonstrating percentageinfection on uncloned and cloned PK15 cell monolayers infected with PCV2on 3 days post-infection. A, B, C and D represent mock-infected PK15monolayer (negative control), infected PK15 monolayer, low-permissiveand high-permissive (clone C1) subcloned monolayers respectively.

FIG. 2 shows PCV2 attachment onto surface membrane of PK15 cell line,low- and high-permissive cell clones after 1 hour adsorption at 37° C.

FIG. 3 shows growth curves of PK15 and clone C1 cell populations over 48hours.

FIG. 4 shows PCV2 virus yields generated in parental PK15 and cloned C1cell lines over 4 passages.

FIG. 5 shows PCV2 virus genome synthesis in parental PK15 and cloned C1cell lines.

SUMMARY OF THE INVENTION

The present invention provides a continuous cell line that is highlypermissive to PCV2 infection. In another embodiment, the inventionprovides a method for producing a substantially homogeneous cell linethat is highly permissive to PCV2 infection, which comprises (1)cultivating a heterogeneous cell population that contains cells ofvarying susceptibility to PCV2 infection; (2) diluting the cell cultureand placing aliquots of the diluted cells into separate vessels suchthat each vessel contains about one cell; (3) adding PCV2 to eachvessel; (4) culturing the cells and identifying a vessel that containscells that are susceptible to PCV2 infection; and (5) culturing andmaintaining a cell line from such susceptible cells. In a particularembodiment, the invention provides a continuous cell line designatedPK15-C1. In yet another embodiment, the invention provides a method forproducing PCV2 by cultivating a virus in a cell line of the presentinvention under conditions suitable for cell growth and recovering virusproduced by the cell line.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention, it has been discovered that thepopulation of cells in the PK15 porcine kidney cell line isheterogeneous with respect to permissivity to the PCV2 infection. Thecell line has been found to contain cells of both low- andhigh-permissivity to viral infection. The relatively low virus titersproduced by PCV2-infected PK15 are attributable to the heterogeneity ofthe cell line.

Homogeneous cell lines of this invention may be produced by cloningsingle cells and identifying resulting cultures that are highlysusceptible to PCV2 infection. While the PK15 cell line is a preferredcell line for use in the methods of this invention, other cell linesthat are susceptible to infection with PCV2 may also be used to producehomogeneous virus-producing cell lines.

A culture of a heterogeneous cell population is first diluted intoaliquots containing single cells. The single-cell aliquots are placedinto individual vessels, such as the wells of a microtiter plate, andare suspended in a nutrient medium that contains nutrients, growthfactors and buffers necessary for replication of the cells. They arethen incubated under thermal and atmospheric conditions conducive tocell growth. Following cell growth, e.g., to a continuous monolayer, aninfectious amount of virus is added to each aliquot and the cells arecultured until a suitable phase of virus production is achieved.

Virus titers of each aliquot are determined, e.g., by immunofluorescenceassay according to the following protocol: Infected cells are fixed with4% paraformaldehyde for 15 minutes at 72 hours post-infection (pi), andincubated with PCV2 ORF-1 antibody followed by anti-guinea pigfluorescein isothyocianate (FITC), each for 1 hr at 37° C. with washingof cells one time with phosphate buffered saline between each step. Thestaining results are observed under an Olympus fluorescent microscopeand cells are scored for their ability to produce high virus titers.High virus-producing cell lines are then advantageously re-cloned andhighly permissive cells are selected.

A preferred cell line produced in accordance with the invention wasderived from cell line PK15 (ATCC® CCL-33™ pig kidney cell line) and hasbeen designated clone C1. It is referred to herein as PK15-C1. Theresults presented herein show that clonal C1 cell population is moresusceptible to PCV2 infection than are PK15 cells. Therefore, PK15-C1 isa more effective cell line for the production of high PCV2 virus yieldthan the parental PK15 cell line.

PK15-C1 has been deposited with the American Type Culture Collectionlocated at University Boulevard, Manassas, Va. 20110, USA on 20 Mar.2007 and assigned accession number PTA-8244.

The invention is further illustrated by the following examples, whichare not intended to limit the invention.

Example 1 Production of PK15-C1

The PK15 parent cell line was maintained in Eagle's minimum essentialmedium (MEM), supplemented with 5% fetal bovine serum (FBS), 2.2 g/Lsodium bicarbonate, 2 mM L-glutamine, 1.0 mM sodium pyruvate andantibiotics. Cloning of PK15 cells was performed by thelimiting-dilution method. The cells were trypsinized, diluted at a meanconcentration of 1 cell/well in MEM/20% FBS/60% conditioned media anddispensed into 96-well tissue culture plates. The wells were immediatelyscreened for single cells and marked, and the plates were incubated at37° C. in an atmosphere of 5% CO₂. Following the identification of cellmonolayers from the initial cloning, these subclones were subjected toanother round of further cloning.

The PK15 parent and cloned cell populations were screened byimmunofluorescence assay (IFA) for high- and low-permissive cells. Thecells were seeded to 70% confluency in 96-well plates and infected withPCV2 with a titer of about 10⁵ TCID₅₀/ml at 6 hours post-seeding.Glucosamine (300 mM) was added to the infected cells within 24 hours ofinfection, and the cells were maintained in MEM/5% FBS at 37° C./5% CO₂.Positive and negative controls used in this experiment were PCV2 virussupernatant (s/n) of 10⁵ TCID₅₀/ml and MEM/5% FBS respectively. Thecells were fixed with 4% paraformaldehyde, and IFA was performed at 72hours post-infection (pi). IFA results demonstrated highest of 90% PCV2infection in high-permissive clone C1 cells, compared to only 40% PCV2infection in PK15 parent cells, and less than 20% infection in theremaining low-permissive cell clones (FIG. 1). In addition, a virusattachment assay was carried out to observe the affinity of PCV2 to thecell surface membrane of each cell clone. Each cell clone was seededonto chamber slides and incubated overnight as above to obtain 70%confluency. PCV2 of 10⁷ TCID₅₀/ml was added to the cells for 1 houradsorption at 37° C. and fixed with 4% paraformaldehyde. IFA wasperformed as above, however using PCV2 ORF-2 antibody for the primaryantibody. After the final washing, the stained cells were mounted withfluorescent mounting media and observed under a Zeiss Meta invertedconfocal microscope. Confocal microscopy results demonstrating PCV2attachment onto cell surface membranes of cloned and uncloned PK15 cellsafter 1 hour adsorption at 37° C. are shown in FIG. 2. A, B, C and Drepresent mock-infected PK15 monolayer (negative control), infectedlow-permissive, high-permissive (clone C1) subcloned monolayers andinfected PK15 monolayer respectively. 1) FITC fluorescent stainingimage. 2) Overlay of light phase and green fluorescence image. Thespread and intensity of fluorescence, indicating PCV2 attachment to thecell surface membrane, was observed most intensely on high-permissivecell clone C1, followed by uncloned PK15 cells and low-permissive cellclones. These results suggested that clone C1 is most susceptible toPCV2 infection, and therefore selected for propagation of PCV2 virus.

Example 2 Characterization of PK15-C1

PK15 parent cells and clone C1 cells were characterized by their meangeneration time in hours. Approximately 1×10⁵ cells were seeded into6-well plates, and cultured in MEM/10% FBS. The cells were trypsinized,counted, and underwent DNA extraction and quantitation at 0, 4, 8, 16,24, 32 and 48 hours post-seeding. From the cell count and DNAquantitation data, the mean generation times of PK15 parent and clone C1cell populations were determined to be 12.1 and 14.6 hours respectively.The results shown in FIG. 3 and Table 1 suggest that PK15 parent cellsdoubles faster than that of clone C1 cells.

Following, the titres for released virus in the culture supernatant werecompared between virus yields from PK15 parent and clone C1 cells. Thecells were seeded to 70% confluency in 150 cm² tissue culture flasks andinfected with PCV2 (10⁴ TCID₅₀/ml) at 6 hours post-seeding. The infectedcultures were treated with D-glucosamine and maintained in culture mediaas previously mentioned. Finally, the virus-infected cultures werefreeze-thawed three times at 4 days post infection (DPI), cells debriswere pelleted at 3500 rpm at 4° C. for 5 minutes and supernatantcontaining PCV2 virus was retrieved. PCV2 virus was serially passaged inPK15 parent and clone C1 cell lines, harvested and stored at −80° C.until infectivity was determined by IFA using C1 clones. IFA resultsdemonstrated that C1 cell clone produced a maximum virus titer of 10⁸TCID₅₀/mL after 5 serial passages compared to a lower titer of 10⁵TCID₅₀/mL generated from the parental PK15 cell line (FIG. 4).

TABLE 1 Cell Line Mean Generation Time (Hours) PK15 12.1 Clone C1 14.6

DNA replication rates of PCV2 in the parental PK15 and C1 cell clonewere also assessed using a real-time PCR method. Two hundred microlitersof each PCV2 infected PK15 and C1 cell lysate were harvested at 4 daypost-infection (DPI) and DNA extractions were carried out using theQiaAmp DNA Mini kit (QIAGEN, Inc., Valencia, Calif. USA). The purifiedDNA was then eluted in 200 microliters of sterile distill water.Real-time PCR was carried out using the Roche LightCycler system (RocheApplied Science, Indianapolis, Ind. USA). One microliter of each DNAextract was used as PCR template and a pair of PCV2 specific primers wasused for the amplification (Forward primer: 5′ cacctggttgtggtaaaagc 3′,Reverse primer: 5′ ggtctgattgctggtaatcg 3′). A PBluescript plasmid(Stratagene, La Jolla, Calif. USA) containing PCV2 genome insert wasused as standard reference. Real-time PCR quantification has shown thatthe genomic DNA copy number of PCV2 in 1 mL of PK15 and C1 cell lysatesare 10⁷ and 10¹⁰ respectively (FIG. 5).

Although the invention has been described herein in detail for thepurpose of illustration, it is to be understood that variations can bemade therein by those skilled in the art without departing from thespirit and scope of the invention except as it may be limited by theclaims.

We claim:
 1. A method for the continuous production of porcinecircovirus type 2 (“PCV2”), which comprises infecting a porcine kidneycell line PK15-C1 with said virus, growing said cell line underconditions suitable for cell growth; and recovering said virus producedby said cell line, wherein said porcine kidney cell line PK15-C1 is thecell line PK15-C1 deposited with the American Type Culture Collection onMar. 20, 2007 as PTA-8244.
 2. The cell line PK15-C1 deposited with theAmerican Type Culture Collection on Mar. 20, 2007, as PTA-8244.
 3. Aporcine kidney cell line PK15-C1 for continually producing porcinecircovirus type 2 (“PCV2”), wherein the cell line is produced by amethod which comprises: (1) cultivating a heterogeneous PK15 cellpopulation that contains cells of varying susceptibility to PCV2infection; (2) diluting the cell culture and placing aliquots of thediluted cells into separate vessels such that each vessel contains aboutone cell; (3) adding PCV2 to each vessel; (4) culturing the cells andidentifying a vessel that contains cells that are more permissive toPCV2 infection; and (5) culturing and maintaining a cell line from themore permissive cells identified in step 4, wherein said cell line fromstep 5 is the cell line PK15-C1 deposited with the American Type CultureCollection on Mar. 20, 2007, as PTA-8244.